We are implementing an approach to improve the efficiency and applicability of insertional mutagenesis as a method to identify and clone a large number of developmentally important genes. This approach is based on a novel multiplex strategy for analyzing several sets of different proviruses, each present at multiple copy number and all integrated into the germ line of the same transgenic mouse strain. Clones of ES cells have been derived by multiple infection with 6 different retroviral vectors and analyzed to identify ones that average 2O copies of each provirus type (and a total copy number of approximately 120). One clone is now being used to make injection chimeras by a new approach that improves efficiency. We use carrier blastocysts derived from super-ovulated C57/B16 mice. We have shown that higher numbers of embryos can be harvested per donor mouse, as expected. But also, we have shown that these embryos do not differ from ones collected from naturally mated females, in terms of viability after transfer to foster mothers.